G proteins function as cellular biological switches, which effectively determine when and for how long signal transduction pathways are activated. The biological switch is activated when GTP (guanosine triphosphate) is bound, and inactivated when GTP is hydrolyzed to GDP (guanosine diphosphate). The rate of hydrolysis determines the duration between biological activation of the signal pathway and deactivation. G protein signal transducers form a superfamily of GTPases. GTPase family members, most notably the products of ras genes, regulate the rate of cell division. Linder, M. E., et al., Scientific American, 56:56 (1992).
Many important cell-surface receptors for hormones and sensory stimuli, for example, transduce extracellular stimuli into cellular responses by promoting formation of the GTP-bound state of their GTPase targets. Each protein in the family is a precisely engineered molecular switch that can change its affinities for other macromolecules. Turned on by binding GTP and off by hydrolysing GTP to GDP, the switch mechanism is remarkably versatile, enabling different GTPases to sort and amplify transmembrane signals, direct the synthesis and translocation of proteins, guide vesicular traffic through the cytoplasm, and control proliferation and differentiation of cells. As targets of mutation and microbial toxins, GTPases have pivotal roles in the pathogenesis of cancer and infectious diseases. Bourne, H. R., et al., Nature, 349:117 (1991).
Ras is a small guanine nucleotide binding GTPase that transduces biological information from the cell surface to the nucleus. Its ability to transfer growth signals from receptor tyrosine kinases to a mitogen activated protein kinase (MAPK) cascade puts it in the heart of signaling pathways that cause proliferation in normal cells and uncontrolled growth in cancer cells. Indeed, mutations that lock Ras in its active, GTP-bound state lead to malignant transformation and are among the most frequently identified mutations in human cancers. Barbacid, M., Annu. Rev. Biochem., 56:779 (1987); McCormick, F., Nature, 363:15-16 (1993). The Ras family of low molecular weight GTP-binding proteins has been implicated in a wide range of cellular processes, including cell growth and differentiation, intracellular vesicular trafficking, nucleocytoplasmic transport, and cytoskeletonal reorganization. Bourne, H. R., et al., Nature, 348:125 (1990); Zerial, M., et al., Guidebook to the Small GTPases, Oxford University Press, New York (1995).
A newly described subfamily of Ras-like GTPases composed of the Rad, Kir, and Gem proteins is now apparent. Finlin, B. S., et al., J. Biol. Chem., 272:35, 21982 (1997). The members of this Ras subfamily are subject to transcriptional regulation. Rad is overexpressed in muscle of type II diabetes patients and Kir/Gem expression is induced by oncogenic kinases (reviewed infra). Kahn, C. R., et al., Science, 262:1441 (1993); Cohen, L. et al., Transcriptional Activation of a Ras-like Gene (Kir) by Oncogenic Tyrosine Kinases, PNAS, 91:12448 (1994). Non-insulin dependent diabetes mellitus (NIDDM, or Type II diabetes) is among the most common metabolic disorders, affecting up to 6% of the population of the United States. The high incidence of diabetes in certain populations and among first-degree relatives of Type II diabetic patients, as well as the high concordance in identical twins, provide strong evidence that genetic factors underlie susceptibility to this disease. Reynet, C., et al., Rad: A Member of the Ras Family Overexpressed in Muscle of Type II Diabetic Humans, Science, 262:1441 (1993). Rad has also been shown to associate with skeletal muscle .beta.-tropomyosin and the cytoskeleton of muscle cells and to inhibit insulin-stimulated glucose uptake in a variety of cultured cell lines. Zhu, J., et al., J. Biol. Chem. 271:2, 768 (1996); Moyers, J. S., et al., J. Biol. Chem., 271:23111 (1996).
The mRNA levels of both Kir and Gem are transcriptionally induced in activated T-lymphocytes. lymphocytes. Finlin, B. S., et al., J. Biol. Chem., 272:35, 21982 (1997). Murine Kir and Gem nucleotide sequences are 98.4% identical in their coding sequence and most likely encode the same protein or very highly related proteins, referred to as Kir/Gem. Cohen, L. et al., Transcriptional Activation of a Ras-like Gene (Kir) by Oncogenic Tyrosine Kinases, PNAS, 91:12448 (1994). Gem, shares significant sequence homology with Rad and is a mitogen-induced immediate early gene product in T lymphocytes. Zhu, J., et al., J. Biol. Chem., 271:2, 768 (1996). Moreover, deregulated expression of Gem has been demonstrated to prevent proliferation of normal and transformed 3T3 cells, clearly suggesting that Gem is involved in regulating signaling pathways that influence cell growth. Maguire, J., et al., Science. 265:241 (1994). Furthermore, cellular levels of Kir are dramatically increased in pre-B cells transformed by a set of abl tyrosine kinase oncogenes. Cohen, L. et al., PNAS, 91:12448 (1994). The correlation between Kir expression and the tumorigenic and metastatic potential of BCL/ABL and v-ABL transformed cells suggests that Kir participates in the processes of metastasis. Genetic analysis moreover suggests that Kir acts upstream of the STE20 kinase and results in the activation of a mitogen-activated protein kinase (MAPK) cascasde. Doreen, D. et al., Oncogene, 11:2267 (1995). These results are consistent with an increasingly accepted model in which members of the Kir/Gem and Rad subfamily, regulate growth-related cellular signaling cascades by controlling the activity of mitogen-activated protein kinases (MAPK). Finlin, B. S., et al., J. Biol. Chem., 272:35, 21982 (1997); Fischer, R., et al., J. Biol. Chem., 271:41, 25067 (1996).
Evidence has demonstrated that MAPK and stress activated protein kinase (SAPK) signal transduction pathways are responsible for triggering biological effects across a wide variety of pathophysiological conditions including conditions manifested by dysfunctional leukocytes, T-lymphocytes, acute and chronic inflammatory disease, auto-immune disorders, rheumatoid arthritis, osteoarthritis, transplant rejection, macrophage regulation, endothelial cell regulation, angiogenesis, atherosclerosis, fibroblasts regulation, pathological fibrosis, asthma, allergic response, ARDS, atheroma, osteoarthritis, heart failure, cancer, diabetes, obeisity, cachexia, Alzheimers disease, sepsis, and neurodegeneration. As MAP kinases play a central role in signaling events which mediate cellular responses, controlling the activity of MAPK is key to the attenuation of the response. N. J. Holbrook, et al., Stress-Inducible Cellular Responses, 273, Feige, U., et al., Eds., Birkhauser Verlag (1996).
Angiogenic response of vascular endothelium, endothelial cell proliferation, is one of the first steps in angiogenesis. VEGF, bFGF, and EGF all upregulate MAP kinase in HUVEC cells. The establishment and remodeling of blood vessels is controlled by paracrine signals, many of which are protein ligands that bind and modulate the activity of transmembrane receptor tyrosine kinases (RTKs). The basic view of RTK signaling has come from studies (performed largely in fibroblasts) of ligand-dependent autophosphorylation and activation of the branched Ras pathways. Results suggest that most RTKs are similarly coupled into the intracellular signal transduction cascade and are capable of inudcing endothelial cell proliferation. Hanahan, D., Signaling Vascular Morphogenesis and Maintenance, =l Science, 227:48 (1997).
Finlin et al. recently reported the cDNA cloning and characterization of a murine GTP-binding protein, Rem (for Rad and Gem-related). Alignment of the full-length open reading frame of mouse Rem revealed the encoded protein to be 47% identical to the Rad, Gem, and Kir proteins. The distinct structural features of the Rad, Gem, and Kir subfamily are maintained including a series of nonconservative amino acid substitutions at positions important for GTPase activity and a unique sequence motif thought to direct membrane association. Recombinant Rem is reported to bind GTP in a specific and saturable manner. The administration of LPS (lipopolysaccharide) to mice, a potent activator of the inflammatory and immune systems, results in the general repression of Rem mRNA levels in a dose- and time-dependent manner. The unique structure of Rem, its enrichment in tissues with a large number of vascular endothelial cells, its ability to specifically bind GTP, and its regulation by LPS suggest that it may control cellular pathways in endothelial cells. Finlin, B. S., et al., J. Biol. Chem., 272:35, 21982 (1997). Finlin, B. S., et al., Rem is a New Member of the Rad- and Gem/Kir Ras-Related GTP-Binding Protein Family Repressed by Lipopolysaccharide Stimulation, J. Biol. Chem., 272:35, 21982 (1997). See also, regarding hypertension, Iiri, T., et al., Nature Genetics, 18:8, et seq (1998).
Compounds which are able to modulate the activity of specific signal transduction molecules integral to specific intracellular pathways are expected to have significant potential for the ability to control or attenuate downstream physiological responses. Unfortunately, in spite of the introduction of numerous new drugs during the last three decades, there is a need for new, more efficient and less toxic compounds. Accordingly, the ability to identify such compounds is of paramount importance.
Rem is an exemplary biological candidate of the newly described subfamily of Ras-like GTPases for angiogenesis regulation as well as regulation of functional physiology in endothelial and smooth muscle cells, rates of cell division, differentiation, as well as peripheral vascular disease, inflammation, arteriosclerosis, hypertension, pathogenesis of cancer and infectious diseases, COPD (chronic obstructive pulmonary disease), leukocyte physiology, T-lymphocyte activity, diabetes, as well as cell activation, shape, and motility, inter alia. However, the previously reported Rem GTPase is a murine isolate. The availability of a functional human homolog will be ideal for such drug screening as well as diagnosis, study, prevention, and treatment of pathophysiological disorders related to the biological molecule.